Thrombin derived polypeptides; compositions and methods for use

ABSTRACT

Thrombin is now known to mediate a number of potent biological effects on cells bearing high-affinity thrombin receptors. These effects depend, at least in part, upon receptor occupancy signals generated by thrombin&#39;s interaction with the high affinity thrombin receptor. The present inventors have formulated synthetic thrombin derivatives capable of selectively stimulating or inhibiting thrombin receptor occupancy signals. The stimulatory thrombin derivatives to bind to cell surface thrombin receptors and stimulate DNA synthesis in cells treated with non-mitogenic concentrations of alpha-thrombin or phorbol myristate acetate. Thus, these peptides, which have both a thrombin receptor binding domain and a segment of amino acids with a sequence common to a number of serine proteases, appear to generate receptor-occupancy dependent mitogenic signals. The inhibitory derivatives, which have no serine esterase conserved amino acid sequences bind to thrombin receptors without generating receptor-occupancy dependent mitogenic signals. This invention describes the peptides and methods for using them to promote cell growth and wound healing or to inhibit scar formation, tissue adhesions, and tumor metastasis and angiogenesis.

This is a continuation of application Ser. No. 08/007,173, filed Jan.21, 1993, now U.S. Pat. No. 5,500,412; which is a divisional ofapplication Ser. No. 06/925,201, filed Oct. 31, 1986, now U.S. Pat. No.5,352,664.

The U.S. Government own rights in the present invention pursuant togrant number CA00805 and AM25807 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to chemical compounds and methodsuseful in the regulation of thrombin receptor mediated cell stimulation.More specifically, the invention is directed to prothrombin-derivedpeptides and methods which employ such peptides for promoting woundhealing and inhibiting scar formation, tissue adhesions, bloodcoagulation, tumor angiogenesis, tumor metastasis and pulmonary edema.

2. Description of the Related Art

Human alpha-thrombin appears to have growth-promoting activity for awide variety of cells from various tissues. For example, alpha-thrombinhas been shown to initiate proliferation of fibroblastic cells inculture without addition of serum or other purified growth factors, tosynergize with epidermal growth factor in certain hamster fibroblastsand human endothelial cells, and to initiate cell division or DNAsynthesis in mammalian lens epithelial and spleen cells. Yet, the use ofthrombin as a growth factor and its potential importance to woundhealing has not been widely acclaimed. In part, this may be due to thecomplexity of thrombin's involvement with coagulation, plateletactivation, and initiation of cell proliferation as well as to thecomplex regulation of thrombin and thrombin-like molecules by serumprotease inhibitors and by cell-released protease nexins. Thiscomplexity and high degree of physiologic regulation, however, supportsthe potential importance of this initiation pathway in wound healing.

Thrombin may also play a role in metastasis and angiogenesis of tumors.Generally, for a tumor to grow larger than a few millimeters indiameter, vascular endothelium must proliferate and form vesicle wallsto provide circulation and nutrients to the cells inside of the tumormass. Thrombin likely potentiates this process by virtue of its abilityto induce proliferation of endothelial cells. In addition, thrombin hasbeen shown to disrupt the normal intercellular endothelial cell contactsimportant in preventing cells and plasma factors from escaping orentering the microvasculature. The present hypothesis that thrombin mayincrease metastasis by disrupting these contacts is supported by studiesdemonstrating a correlation between decreased levels of anti-thrombinIII (which removes thrombin and other proteases from plasma) andincreased tumor metastasis.

Various studies have led the present inventors to conclude thathigh-affinity cell surface thrombin receptors (See Carney andCunningham, Cell 15:1341, 1978) may be involved in tumor metastasis andangiogenesis. For example, studies have indicated that thrombinreceptors can serve as binding sites for tissue plasminogen activator, amolecule secreted from metastatic tumor cells. Moreover, other studiesdemonstrate the involvement of tissue plasminogen activator inmetastasis and angiogenesis. Thus, many of the effects of plasminogenactivator may be mediated through its interaction with the cell surfacethrombin receptor. It is therefore proposed that stimulation of thethrombin receptor serves to promote tumor metastases, while inhibitionof the receptor will decrease metastatic activity.

Thrombin has also been shown to cause changes in the structure andfunction of cells which make up the endothelial vasculature. Thesestudies suggest that thrombin may play a central role in the developmentof pulmonary edema as well as edema of other tissues. For example,thrombin has been shown to increase permeability of endothelial cellmonolayers to macromolecules, to increase arterial pressure andpulmonary vascular resistance, to induce smooth muscle contraction, andto increase transcapillary fluid filtration. All of these effects may bemediated by thrombin's interaction with cell surface thrombin receptors.

A number of recent studies have attempted to elucidate the mechanismsfor thrombin-mediated cell stimulation. These studies have indicated tothe present inventors that initiation of cell proliferation by thrombinrequires two signals. The first signal appears to be dependent uponbinding of the thrombin molecule to the high affinity cell surfacethrombin receptor, while the second signal results from the enzymicactivity of the thrombin molecule. Thus, unlike alpha-thrombin, neitherDIP-alpha-thrombin (a proteolytically inactive thrombin derivative thatretains receptor-binding activity) nor gamma-thrombin (anesterolytically active, but non-binding thrombin derivative) caninitiate DNA synthesis or cell division. However, simultaneous additionof these two non-mitogenic thrombin derivatives initiates a level of DNAsynthesis and cell division comparable to that initiated byalpha-thrombin.

These same thrombin derivatives have been used to distinguishintracellular events stimulated by high-affinity thrombin receptoroccupancy from those resulting from proteolytic cleavage. Alpha-thrombinand gamma-thrombin both stimulate Na⁺/K⁺ ATPase activity,phosphoinositol turnover, and Ca²⁺ metabolism, whereasDIP-alpha-thrombin does not. Thus, these events are attributable tothrombin's enzymic activity, not to receptor occupancy. Furthermore,these signals (the release of diacylglycerol and inositol triphosphateto cause Ca²⁺ mobilization) may in turn activate protein kinase C.Accordingly, it has been shown that phorbol myristate acetate (PMA),which activates protein kinase C, can substitute for enzymically activegamma-thrombin and initiate cell division in the presence of receptorsaturating levels of DIP-alpha-thrombin or monoclonal antibody to thethrombin receptor. Thus, the requirements for enzymically activethrombin may indirectly relate to its activation of protein kinase C.

The precise signals generated by high-affinity interaction of thrombinwith its receptor have been more difficult to define. However, it hasrecently been shown that DIP-alpha-thrombin stimulates a transientincrease in intracellular cAMP. In contrast to ion fluxes andphosphoinositide turnover, cAMP levels are maximally stimulated byDIP-alpha-thrombin but are not stimulated by gamma-thrombin. Attempts toreplace DIP-alpha-thrombin with cAMP analogs, however, have beenunsuccessful. Therefore, it is possible that thrombin receptor occupancyproduces a number of signals in addition to changes in cAMP levels.

One problem associated with the clinical application of thrombindirectly to achieve such benefits is its susceptability to proteaseinhibitors by serum anti-thrombins. Such problems have heretoforeprevented the use of thrombin in the clinic and has led the presentinventors to identify smaller thrombin-active and thrombin antagonisticpolypeptides which are not sensitive to the inhibitory effects ofthrombin inhibitors.

The present invention provides for a number of smaller polypeptideswhich have been tailored to interact with the thrombin receptor toselectively stimulate or inhibit thrombin receptor occupancy relatedsignals. It is believed that these polypeptides will prove to be usefulin a wide variety of clinical settings where successful recovery may beinfluenced by thrombin receptor-mediated events.

SUMMARY OF THE INVENTION

The present invention provides a number of thrombin derivatives andmethods useful for stimulating cell proliferation and promoting woundhealing as well as methods useful in inhibiting wound healing, scartissue formation, formation of tissue adhesions, and tumor metastasisand angiogenesis. The invention is based on the discovery that one mayformulate polypeptide thrombin derivatives, or their physiologicallyfunctional equivalents, which selectively inhibit the interaction ofthrombin with its high-affinity receptor or which mimic the stimulatoryeffects of thrombin.

Accordingly, the present invention, in its most general and overallscope, relates to synthetic or naturally derived polypeptide agonistsand antagonists of thrombin receptor mediated events. Both of theseclasses of agents possess a thrombin receptor binding domain whichincludes a segment of the polypeptide that is capable of selectivelybinding to the high-affinity thrombin receptor. This segment of thepolypeptide includes a sequence of amino acids homologous to atripeptide cell binding domain of fibronectin.

In addition to the thrombin receptor binding domain, the stimulatory(agonistic) polypeptides possess a sequence of amino acids havingsequences derived from the N-terminal amino acids of a dodecapeptidepreviously shown to be highly conserved among serine proteases. However,the inhibitory polypeptides do not include these serineesterase-conserved sequences.

The present invention is disclosed in terms of a showing that in thepresence of a non-mitogenic (ie, non-stimulatory) concentration ofalpha-thrombin, gamma-thrombin, or PMA, the interaction betweenstimulatory polypeptides and cell surface thrombin receptors providesthe cell with a signal to proliferate. However, no proliferative signalresults when cell surface thrombin receptors interact with theinhibitory polypeptides. Instead, the cells become more refractory tosubsequent treatment with the stimulatory polypeptides. This result isbelieved to occur because the inhibitory polypeptides, which arethemselves incapable of generating a proliferative signal, block bindingof the stimulatory polypeptides.

As indicated above, practice of the cell-stimulatory methods of thepresent invention requires the presence of a secondary signal, forexample, in the form of non-mitogenic concentrations of alpha-thrombin,gamma-thrombin, or PMA in order to supply the cells with thelow-affinity proteolytic cleavage signal. Accordingly, the inventionprovides for pharmaceutical compositions and methods to which thesecompounds have been added. However, those of skill in the art willrecognize that when the invention is practiced in vivo, nativealpha-thrombin endogenous to the host will typically be adequate toprovide this secondary signal.

Because thrombin is involved in a number of bioregulatory effects, thepresent invention, which allows one to selectively promote and inhibitthese effects, has a number of clinical applications. For example, theinvention provides a number of polypeptides useful in promoting woundhealing. For such applications, the invention provides a polypeptidederivative of thrombin (or a functional equivalent of such a derivative)which has a thrombin receptor binding domain as well as a domain with aserine esterase conserved sequence of at least 12 amino acids. Theinvention also provides a polypeptide compound of at least 23 L-aminoacids which has both. a thrombin receptor binding domain and a domainwith a serine esterase conserved amino acid sequence.

In one embodiment, the invention provides for several polypeptidescontaining specific amino acid sequences, such as a polypeptide compoundin which the thrombin receptor binding domain includes the L-amino acidsequence Arg-Gly-Asp-Ala (SEQ ID NO: 1) together with the serineesterase conserved amino acid sequence,Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO:2). In apreferred embodiment, the polypeptide compound includes the L-amino acidsequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO:3).

The invention also provides for a pharmaceutical composition forpromoting wound healing which includes of a therapeutically effectiveconcentration of any of the compounds described above combined with apharmaceutically acceptable excipient. Typically, such compositionsinclude, for example, sufficient concentrations of the polypeptides toeffect a stimulatory action on the thrombin receptor as demonstratedherein. Thus, such compositions should typically include sufficientconcentrations to obtain levels of the polypeptides in the wound areawhich are shown in vitro to stimulate the receptor. When endogenouslevels of a secondary signal are believed to be inadequate, compositionsmay be employed which further include the addition of a therapeuticallyeffective concentration of alpha-thrombin or gamma-thrombin.

As used herein, a therapeutically effective concentration is defined asa concentration of the particular agent which provides a satisfactoryincrease in the rate of wound healing. Again, such concentrations arebelieved to correspond to levels sufficient to ellicit a stimulation ofthe thrombin receptor in vitro. However, it is believed that thecompositions will prove most effective when the stimulatory (agonistic)polypeptides are present at a concentration of from 0.1 uM to 10 uM.

Furthermore, where alpha-thrombin or gamma-thrombin are also employed,concentrations of from 0.1 uM to 10 uM are considered effective.However, empirical methods as are known in the art may be employed fordetermining more precisely the proper therapeutic dose for a givencomposition administered in a particular manner.

In addition, methods are provided which employ thrombin agonists topromote wound healing. One such method includes applying to the wound atherapeutically effective amount of a polypeptide derivative ofthrombin, or a physiologically functional equivalent thereof, which hasboth a thrombin receptor-binding domain and a domain having a serineesterase conserved amino acid sequence. In general, thrombin is appliedin amount sufficient to achieve fibroblast stimulation and therebystimulate tissue regeneration. In that such methods typically involvetopical application to a wound, possible sytstemic toxicity is notbelieved to be a problem. Therefore, virtually any concentration may beemployed. However, in a preferred embodiment, the wound is treated toachieve a range of approximately 1 ng/cm²-10 ug/cm² of wound surface.

The invention further provides a method for promoting wound healing inwhich a therapeutically effective amount of alpha-thrombin (1 ng/cm²-10ug/cm² of wound surface) or gamma-thrombin (1 ng/cm²-10 ug/cm² of woundsurface) is applied tolthe wound in conjunction with the foregoingthrombin derivatives. Of course, the specific polypeptides andpharmaceutical compositions provided by the invention may also be usedin promoting wound healing. It is believed that these methods will beespecially beneficial to patients involved in severe accidents(particularly burn patients), to those subjected to surgical proceduresand to those with poor wound healing responses, such as aged anddiabetic individuals.

Additional methods are provided for using the thrombin receptorinhibitory polypeptides. For example, the invention provides methodswhereby scar tissue formation can be inhibited by administering to thewound or scar tissue, a therapeutically effective amount of apolypeptide derivative of thrombin, or a physiologically functionalequivalent thereof, which has a thrombin receptor binding domain butdoes not have a serine esterase conserved sequence. Typically, suchconcentrations are adequate when sufficient to inhibit thrombin receptormediated events. In a preferred embodiment, amounts ranging from 1ng/cm²-10 ug/cm² of wound surface are considered appropriate.

In a preferred embodiment, the polypeptide derivative of thrombin hasthe L-amino acid sequence Arg-Gly-Asp-Ala (SEQ ID NO:1).

In general, these methods may be used in any situation where scarformation is undesirable, such as on burn patients or those subjected toopthalmic surgery. Moreover, the methods may also be of use inpreventing keloidal scar formation. It is anticipated that spraying thewound with an aerosol spray will be a particularly sterile andefficacious manner of administering the polypeptide compound to thewounds of burn patients.

The inhibitory polypeptides should also prove useful in inhibiting theformation of tissue adhesions, defined as abnormal unions between bodyorgans by formation of fibrous tissue. It is known that fibroblastproliferation is required for formation of such adhesions. Sincealpha-thrombin is known to induce fibroblast proliferation, it followsthat inhibition of thrombin-mediated mitogenesis by the peptides of thepresent invention could reduce adhesion formation. It is believed thatadministration of such inhibitory polypeptides to the surface of theaffected organs will prove to be especially useful following certainsurgical procedures, such as thoracic surgery, where gut adhesions oftenlead to postoperative complications.

It is further proposed that the inhibitory peptides will prove useful inthe treatment of mammals with tumors to thereby inhibit tumor metastasisor angiogenesis. This view is supported by studies demonstrating thatalpha-thrombin is able to disrupt normal inter-endothelial cell contactsimportant in preventing metastasis, as well as studies demonstratingthat alpha-thrombin can induce the proliferation of endothelial cellsrequired for angiogenesis. Accordingly, the invention provides a methodwhereby mammals with such-tumors receive a therapeutically effectiveamount of a polypeptide derivative of thrombin, or a functionalequivalent thereof, which has a thrombin receptor binding domain butdoes not have a serine esterase conserved sequence. While exact doseswould need to be determined by empiracal methods known those skilled inthe art, it is estimated that an amount sufficient to achieve aconcentration of from 0.1 uM to 10 uM at the site to be treated isneeded. Use of a polypeptide wherein the thrombin binding domain has anL-amino acid sequence Arg-Gly-Asp-Ala (SEQ ID NO: 1) is specitfcallyprovided. It is contemplated that the polypeptides will be mostefficacious in this regard when administered intravenously. However,other methods of administration will also likely prove to be effective.

In a most general embodiment, the invention provides for the use ofinhibitory polypeptides to inhibit cell proliferation. This methodencompasses, but is not limited to, situations in which one desires toinhibit cell proliferation in vitro. Of course, the inhibitorypolypeptide, having a thrombin binding domain with the specific sequenceArg-Gly-Asp-Ala (SEQ ID NO: 1), may also be used as a general inhibitorof cell proliferation.

In another general embodiment, the invention comprises methods whereinthe stimulatory polypeptides are used to potentiate cell growth. Apolypeptide including the sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO:3) is specifically provided. This method encompasses, but isnot limited to, situations wherein one wishes to potentiate cell growthin vitro. Such cell-stimulatory uses may be potentiated by furtherproviding an effective amount of alpha-thrombin (0.1 ug/ml-10 ug/ml),gamma-thrombin (0.1 ug/ml-10 ug/ml) or phorbol myristate acetate (10ng/ml-100 ng/ml) in conjunction with the stimulatory polypeptide.

GLOSSARY

For purposes of the present invention, a thrombin derivative is definedas any molecule with an amino acid sequence derived at least in partfrom that of thrombin, whether synthesized in vivo or in vitro.Accordingly, a thrombin derivative, as referred to herein, designates apolypeptide molecule which comprises fewer amino acids than thrombin.

A physiologically functional equivalent of a thrombin derivativeencompasses molecules which differ from thrombin derivatives inparticulars which do not affect the function of the thrombin receptorbinding domain or the serine esterase conserved amino acid sequence.Such particulars may include, but are not limited to, conservative aminoacid substitutions and modifications, for example, amidation of thecarboxyl terminus, acetylation of the amino terminus, conjugation of thepolypeptide to a physiologically inert carrier molecule, or sequencealterations in accordance with the serine esterase conserved sequences.

A thrombin receptor binding domain is defined as a polypeptide sequencewhich directly binds to the thrombin receptor and/or competitivelyinhibits binding between high-affinity thrombin receptors andalpha-thrombin.

A domain having a serine esterase conserved sequence comprises apolypeptide sequence containing at least 4-12 of the N-terminal aminoacids of the dodecapeptide previously shown to be highly conserved amongserine proteases (Asp-X₁-Cys-X₂-Gly-Asp-Ser-Gly-Gly-Pro-X₃-Val (SEQ IDNO:4)). wherein X₁ is either Ala or Ser; X₂ is either Glu or Gln; and X₃is either Phe, Met, Leu, His, or Val).

A stimulatory polypeptide is defined as a polypeptide derivative ofthrombin, or a physiologically functional equivalent thereof, having theability to both bind to and stimulate the thrombin receptor. Therefore,the stimulatory peptides will include both a thrombin receptor bindingdomain and a domain with a serine esterase conserved amino acidsequence.

An inhibitory polypeptide is defined as a polypeptide derivative ofthrombin, or a physiologically functional equivalent thereof, having athrombin receptor binding domain but without a serine esterase conservedamino acid sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Computer assisted analysis of the hydropathy, solubility, andpredicted secondary structure for residues 489 to 548 of humanprothrombin. FIG. 1A, hydropathy profile; FIG. 1B, solubility profile;FIG. 1C, predicted tendency for flexible turn; FIG. 1D, predictedtendency for alpha-helix and beta-sheet structure.

FIG. 2. Three-dimensional representation of X-ray crystallographic dataof trypsin with the following PROTEUS computer-assisted substitutions ofthrombin-specific residues: Gly₁₈₇ Lys; Lys₁₈₈ Arg; Ser₁₉₀ Ala; Gln₁₉₂Glu; and Val₁₉₉ Phe is shown in FIG. 2A. FIG. 2B and 2C show only thethree active site residues (His₅₇, Asp₁₀₂, Ser₁₉₅) and residues 183 to200 of trypsin that are located in the homologous region as thrombin'sresidues 510 to 530. These peptides are oriented in the same position asin the rotated model in FIG. 2A.

FIG. 3. Inhibition of [¹²⁵I]-alpha-thrombin binding to mouse embryo (ME)cells by synthetic peptide p508-530. Specific binding of 0.3 nM[¹²⁵I]-alpha-thrombin to ME cells in the presence of the indicatedconcentration of peptide was measured as described in the description ofthe preferred embodiments.

FIG. 4 Effect of p508-530 on [³H]-thymidine incorporation alone or incombination with low concentrations of alpha-thromnbin. Quiescentserum-free cultures of ME (FIG. 4A) or NIL (a hamster fibroblast cellline; FIG. 4B) were treated with the indicted concentrations of p508-530alone (O) or in combination with concentrations of alpha-thrombin whichgave approximately one third of the maximal response; 2 nM for ME cells(FIG. 4A) and 4 nM for NIL cells (FIG. 4B). [³H]-thymidine incorporationwas determined after 24 hours as described in the description of thepreferred embodiments.

FIG. 5 Effect of p508-530 on [³H]-thymidine incorporation in combinationwith PMA. Quiescent cultures of NIL cells were incubated with p508-530alone (O) or in combination with 25 ng/ml PMA. [³H]-thymidineincorporation was determined as described in the description of thepreferred embodiments.

FIG. 6. Comparison between effects of peptides on thrombin-stimulatedthymidine incorporation. Quiescent cultures of NIL cells were incubatedwith increasing concentrations of p508-530, p519-530, or p517-520 in thepresence of 1 nM alpha-thrombin (a marginally mitogenic concentration).Data are expressed for each concentration as a percentage of the effectof alpha-thrombin alone.

FIG. 7. Effect of p517-520 on thrombin stimulation of [³H]-thymidineincorporation. Quiescent cultures of ME cells were incubated withincreasing concentrations of alpha-thrombin alone, or in combinationwith 625 nM p517-520. [³H]-thymidine incorporation was determined asdescribed in the description of the preferred embodiments.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Thrombin, a molecule once considered important only in the context ofblood coagulation, is now shown to mediate a number of potent biologicaleffects not directly related to coagulation. Many of these effects aredue, at least in part, to signals generated by the interaction betweenthrombin or thrombin-like molecules and the high-affinity thrombinreceptors present on the surface of many cells.

Studies performed in connection with the present invention suggestedthat selective regulation of thrombin-mediated events might be achievedthrough the formulation and synthesis of polypeptides specificallydesigned to either mimic or inhibit such events. Development of smallprotease inhibitor resistant polypeptides capable of performing thesefunctions was particularly desirable in view of the susceptibility ofthrombin to proteolytic enzyme inhibitors, such as anti-thrombin.

A number of peptides based on the sequence of human prothrombin weresynthesized and tested for their ability to bind to the receptor and togenerate proliferative signals. The choice of peptides focused on theamino acid sequence of the region of thrombin around its active siteserine. This region contains a domain (represented by residues 517-520of human prothrombin) with a sequence homologous to the tripeptide cellbinding domain of fibronectin, [Arg-Gly-Asp]. This tripeptide sequenceis common to a number of proteins that may interact with cells (reviewedby Rouslahti and Peirschbacher, Cell, 44:517-518 (1985)). Moreover, ithas been shown that a peptide representing 517-520 of human prothrombin(p517-520) and peptides representing 516-522 and 510-526 of humanprothrombin (p516-522 and p510-526, respectively) are able to promotefibroblast attachment comparable to that induced by fibronectin-specificpeptides.

The selected region also possesses a domain (represented by residues519-530 of human prothrombin) with a high degree of homology to a numberof serine esterases.

The present inventors have discovered that a synthetic peptidecontaining both fibronectin- and serine protease-homologous domains(residues 508 to 530 of human prothrombin) binds to thrombin receptorswith high-affinity and substitutes for DIP-alpha-thrombin as aninitiator of receptor occupancy-related mitogenic signals. In contrast,a synthetic peptide containing only the fibronectin-homologous domain(p517-520) binds to the thrombin receptor without inducing mitogenesis.An intermediate peptide (p519-530) retains the ability to mediatemitogenesis but to a much lesser degree than p508-530.

EXAMPLE 1 Selection of Domains of Human Alpha-Thrombin Involved inBinding of Thrombin to Its High Affinity Receptor

To help select peptide sequences that might be involved in receptorbinding, computer analysis was used to predict the overall hydropathy,solubility, and secondary structural features for the 60 amino acidresidues around the active site serine of alpha-thrombin based on thesequence of human prothrombin (Degen et al., Biochein., 22:2087-2097(1983)). As shown in FIGS. 1A and 1B, this region appears to be highlyhydrophilic and soluble, especially near the region that is homologousto fibronectin's cell attachment domain, residues 517 to 520. Analysisof secondary structural features indicated that the region of thrombinfrom residues 511 to 526 has a strong tendency for being a flexible turnregion with very little tendency towards either alpha-helical orbeta-sheet structures (FIGS. 1C and 1D). Taken together, the variouscomputer-assisted analyses strongly suggest that this region of thrombinshould be externally accessible and, therefore, available forinteraction with the thrombin cell surface receptor. Moreover, theregion of thrombin homologous to the cell attachment domain offibronectin is located at or very near the middle of thishydrophilic-flexible turn of thrombin.

Using the three dimensional x-ray crystallographic data for trypsin(Marquart et al., Acta. Crystallogr., 39:480 (1983)), and makingappropriate amino acid substitutions to reflect the thrombin sequencearound the active-site serine portion of trypsin, computer graphicanalysis predicted that residues 510 to 530 of thrombin are locatedalong the edge of the pocket that leads to the active site cleft (FIG.2). In agreement with the predictions of secondary structure discussedabove, amino acid residues 517 to 520 of thrombin are located at theouter most corner of this region of the proposed trypsin/thrombinstructure. Thus, it appeared reasonable that this region of thrombincould be involved in binding to its receptor.

EXAMPLE 2 Synthesis of Peptides

Peptides were synthesized by the solid-phase method (Erickson andMerrifield, The Proteins, 2:255-257, (1976)) using automaticinstrumentation (Applied Biosystems Peptide Synthesizer Model 430A) andpurified by HPLC (Beckman) on a C-18 column eluted with a linearacetonitrile gradient containing 0.5% (v/v) TFA (trifluoroacetic acid).

EXAMPLE 3 Demonstration that the Thrombin Derivatives Selectively Bindto the High-Affinity Thrombin. Receptor

This example demonstrates that the peptides of the present invention areable to selectively bind to the high-affinity thrombin receptors presenton the surfaces of many cell types. In the present embodiment, thisactivity was demonstrated by showing that the peptides of the presentinvention competitively inhibited binding of [¹²⁵ I]-alpha-thrombin tothrombin receptors present on two strains of cultured fibroblasts.Accordingly, the specific techniques described below represent the bestmode for demonstrating this activity known to the inventors at thepresent time.

a. Culture of Fibroblasts Having High-Affinity Thrombin Receptors

As stated above, fibroblasts derived from two sources were used todemonstrate binding of the peptides of the present invention tohigh-affinity thrombin receptors. These cell lines were prepared asfollows:

Primary cultures of fibroblasts were prepared from 9-to 13-day oldembryos of NIH-swiss outbred mice as described by Carney and Cunningham,Cell, 15:1341-1349, (1978). NIL cells, an established strain of hamsterfibroblasts, were maintained as stock cultures and subcultured everyfour days. All cells were grown in Dulbecco-Vogt modified Eagle's (DV)medium supplemented with 10% (v/v) bovine calf serum (CS), in ahumidified atmosphere of 5% CO₂ in air at 37° C.

Quiescent cultures were prepared by subculturing stock cells from 100 mmdishes, using 0.05% (w/v) trypsin and 0.02% EDTA (w/v) inphosphate-buffered saline (PBS) and plating them in 24-well cultureplates in DV medium supplemented with 10% (v/v) CS at 6×10⁴ cells/cm².After allowing the cells to attach overnight, the medium was removed andthe cells were rinsed with DV medium containing no serum. The cells wereincubated in this serum-free medium for 48 hours before the indicatedexperiments. This procedure has been shown to provide nonproliferatingpopulations of mouse and NIL cells that are 90-95% arrested at the G₁/G₀cell cycle interface.

b. Assay for Measurement of Specific Binding of Thrombin and ThrombinDerivatives to the Cell Surface Thrombin Receptor

As stated above, in the present embodiment, thrombin receptor specificbinding activity of the thrombin derivatives was measured as a functionof their ability to competitively inhibit binding between native[¹²⁵I]-thrombin and the thrombin receptor. Specific techniques wherebythe competitive binding studies were performed are set out below.

Human alpha-thrombin was iodinated in the presence of benzamidine (anactive-site competitive inhibitor), lactoperoxidase, and Na[¹²⁵I]. Aftergel filtration and dialysis, the [¹²⁵I]-alpha-thrombin had a specificactivity of 1 to 3×10⁻⁷ CPM/ug and co-migrated with unlabeledalpha-thrombin as a single band on sodium dodecyl sulfate (SDS)polyacrylamide gels. These iodinated preparations retained approximately80% of their fibrinogen clotting activity.

The ability of the synthetic peptides to compete for specific[¹²⁵I]-alpha-thrombin binding to fibroblasts was measured onnonproliferating, mitogenically responsive cultures in 24 well plates(Falcon) at a cell density of approximately 5×10⁴ cells/cm² aspreviously described (Carney and Cunningham, Cell, 15:1341-1349 (1978)).The medium on the cells was changed to binding medium (serum-free DVmedium containing 0.5% (w/v) bovine serum albumin buffered with 15 mMHEPES at pH 7.0). The cells were equilibrated at 23° C. for 30 minutes,and the medium was changed to binding medium containing[¹²⁵I]-alpha-thrombin (10 ng/ml) with the indicated concentrations ofthe peptides. After 2 hours at 23° C., the assay was terminated byquickly rinsing the cells four times with ice-cold PBS. The cells weredissolved in 1 ml of 0.5 N NaOH and the total radioactivity was measuredusing a Beckman gamma counter. Nonspecific binding was measured as theradioactivity bound to cultures after incubation in binding mediumcontaining a 100-fold excess of unlabeled alpha-thrombin. Specificbinding was calculated by subtracting nonspecific binding from totalradioactivity bound to cultures.

C. Thrombin Binding Activity of Selected Thrombin Derivatives

In order to demonstrate the thrombin receptor binding activity of thepolypeptides of the present invention, the peptides synthesized asdescribed in Example 1 were tested for thrombin receptor activity usingthe assay system described immediately above.

More specifically, in order to demonstrate that p508-530 bound tothrombin receptors, confluent cultures of ME cells were incubated with0.3 nM [¹²⁵ I]-alpha-thrombin and concentrations of p508-530 rangingfrom 8 to 4000 nM for 90 minutes at 23° C. A s shown in FIG. 3, p508-530competed for 30% to 70% of the specific binding of [¹²⁵I]-alpha-thrombinto ME cells. Scatchard analysis of the direct binding of [¹²⁵I]-labeledp508-530 indicated a K_(D) of approximately 6×10⁻⁸ M (data not shown).In addition, the specific binding of [¹²⁵I]p508-530 to ME cells could bedisplaced by both excess p508-530 or excess human alpha-thrombin. Thus,it appears that the competit ion of p508-530 for [¹²⁵I]-alpha-thrombinbinding represents the binding of p508-530 to the same sites asalpha-thrombin, but with an affinity approximately one order ofmagnitude lower.

Furthermore, in order to show that the binding and mitogenicactivity ofp508-530 was specific, two synthetic peptides with physical propertiessimilar to p508-530 but no sequence homology to human alpha-thrombinwere tested for their binding properties. Both of these peptides [onewith 12 amino acids (33% hydrophobic residues and a net charge of −3)and a second with 18 amino acids (39% hydrophobic residues and a netcharge of 0] inhibited binding of [¹²⁵I]-alpha-thrombin less than 5% atconcentrations up to 5 uM.

To further identify regions of thrombin involved in high-affinitybinding and generation of mitogenic signals, two peptides representingspecific domains within p508-530 were tested. The first peptiderepresented residues 519 to 530 of the B-chain region of humanprothrombin, a region of thrombin that is highly conserved among serineproteases. The second peptide represented residues 517 to 520 ofprothrombin, a region of thrombin homologous to the fibronectin cellbinding domain.

Both of these peptides were able to compete for 30% to 50% of thebinding of [¹²⁵ I]-alpha-thrombin to ME cells, but both required higherconcentrations than was required with the initial peptide p508-530(Table 1). For example, 30% inhibition of [¹²⁵I]-alpha-thrombin bindingrequired 33- to 50-fold higher concentrations of p519-530 and p517-520than p508-530, respectively. Thus, both of these peptides appear tointeract with thrombin receptors, but at a lower affinity than p508-530.Because p517-520 is homologous to the fibronectin cell binding domain, apeptide having the sequence Ar-Gly-Asp-Ser (SEQ ID NO:1) the sequence ofthe fibronectin specific peptide) was also tested for its ability tocompete for [¹²⁵I]-alpha-thrombin binding. At a concentration of 1.3 uM,this peptide did not compete with [¹²⁵I]-alpha-thrombin for binding.Thus, the receptor for alpha-thrombin is not the same membrane proteinthat specifically interacts with fibronectin and causes the apparentgrowth promoting action of fibronectin. In addition, these resultsdemonstrate the requirement for alanine within the thrombin receptorbinding domain, since substitution of alanine with serine eliminated theability of the synthetic peptide to compete for alpha-thrombin binding.

TABLE 1 Comparison of Peptide Competition for [¹²⁵I]- Alpha-ThrombinBinding to ME Cells. Concentration Maximal % Amino Acid Required forInhibition Peptide Sequence 30% Inhibition (and Conc.) p508-530AGYKPDEG-  6 nM 78% -KRGDACE-   (40 nM) -GDSGGPFV (SEQ ID NO: 3)p519-530 DACEGD- 200 nM 51% -SGGPFV  (800 nM) (SEQ ID NO: 2) p517-520RGDA 300 nM 50% (SEQ ID NO: 2)  (2.7 uM)

Various concentrations of peptides and [¹²⁵I]-alpha-thrombin (1 nM) wereincubated with quiescent ME cells for 90 minutes at 23° C. Specificbinding of [¹²⁵I]-alpha-thrombin was defined as described in Example 3.

EXAMPLE 4 Stimulation of DNA Synthesis by Selected Thrombin Derivatives

This example demonstrates that binding between stimulatory (agonistic)polypeptides and thrombin receptors generates a receptor occupancysignal which induces DNA synthesis and cell division. In the presentembodiment, DNA synthesis and cell proliferation was measured as afunction of [³H] thymidine uptake by cultured fibroblasts exposed toselected polypeptides in the presence of non-mitogenic concentrations ofalpha-thrombin or PMA. Although the in vitro techniques described belowrepresent the best mode for demonstrating the stimulatory activity ofthe selected polypeptides, those skilled in the art will appreciate thatthe principles demonstrated in the in vitro system described immediatelybelow are also applicable in vivo.

a. Techniques for Measuring DNA Synthesis

The effects of the synthetic peptides on DNA synthesis were determinedby measuring the incorporation of methyl-[³H]-thymidine (TdR, ICNPharmaceuticals, Irvine, Calif.) during a 2 hour incubation generallyfrom 22 hours after addition of peptides and/or thrombin (Stiernberg etal., J. Cell Physiol., 120:209-285 (1984)). After incubation, the cellswere extracted and rinsed with ice-cold 10% (w/v) trichloroacetic acid(TCA). The acid precipitable material was dissolved overnight in 0.5 ml0.5 N KOH at 23° C. HCl (1 N), 0.25 ml, was added and the solution wascounted in 10 ml of RediSolv-HPb (Beckman Instruments, Houston, Tex.)scintillation fluid.

b. Mitogenic Activity of Selected Thrombin Derivatives

Each of the thrombin derivatives synthesized was tested for mitogenicactivity as were the two non-thrombin peptides described in Example3(c). The results of these experiments are described below.

The present inventors first tested the ability of p508-530 to stimulateDNA synthesis in non-proliferating cultures of ME or NIL cells. As shownin FIG. 4, p508-530, by itself, was not sufficient to stimulate[³H]-thymidine incorporation into DNA. However, in combination with 2 nMalpha-thrombin, 0.1 uM p508-530 stimulated a 6- or greater than 2-foldincrease in incorporation of [³H]-thymidine into DNA in ME cells whencompared to parallel cultures left untreated or treated withalpha-thrombin alone, respectively (FIG. 4A). A similar mitogenicstimulation was also observed in NIL hamster cells, although it requireda slightly higher concentration of thrombin and peptides (FIG. 4B). Theresponses in both cell types were equivalent to the mitogenic responsestimulated by a maximally effective concentration of alpha-thrombin (10nM). It is noteworthy that for ME cells, stimulation by p508-530 wasobserved between 12.5 nM and 100 nM (FIG. 4A), concentrations thatcorrespond closely with those required to inhibit [¹²⁵I]-alpha-thrombinbinding to ME cells (FIG. 3). With NIL cells, a similar correlation wasobserved between the mitogenic concentrations of p508-530 and theconcentrations required to inhibit thrombin binding, although at higherlevels than required with ME cells.

Although these results suggest that p508-530generates mitogenic signalsthrough its interaction with high-affinity thrombin receptors, it waspossible that the peptide merely increased the effective concentrationof alpha-thrombin. Recently, phorbol myristate acetate (PMA) has beenshown to mimic the effects of gamma-thrombin and stimulate DNA synthesisand cell proliferation in combination with DIP-alpha-thrombin or withmonoclonal antibodies to the thrombin receptor. It was predicted,therefore, that if p508-530 was generating a receptor occupancy-relatedsignal, its addition to cells in combination with PMA should stimulatemitogenesis. As shown in FIG. 5, in the presence of 25 ng/ml PMA (whichis a non-mitogenic amount), p508-530 stimulated a 3.5-fold increase inDNA synthesis over controls. This stimulation occurred at approximatelythe same concentration of peptide as that required to stimulate DNAsynthesis in the presence of low concentrations of alpha-thrombin. Sinceactive thrombin was not present in these experiments, it would appearthat p508-530 itself generates a mitogenic signal that mimics the effectof DIP- or alpha-thrombin binding to high-affinity thrombin receptors.

In order to ensure that the stimulation of DNA synthesis by p508-530 wasmediated by virtue of its ability to interact with the high-affinitythrombin receptor, the synthetic, non-thrombin, non-receptor bindingpolypeptides described in Example 3(c) were tested for initogenicactivity. Neither of these peptides generated a mitogenic response inthe presence of 1 nM alpha-thrombin. Thus, neither the binding activitynor the mitogenic activity of p508-530 is due to non-specificinteraction of the polypeptide with the cells.

The inventors then tested the mitogenic activity of the smaller thrombinderivatives, p519-530 and p517-520. As indicated in Example 3(c) above,both of these peptides bind to the high-affinity thrombin receptor. Inthese experiments, increasing concentrations of p519-530 and p517-520were added to quiescent NIL cells in the presence of 2 and 4 nMalpha-thrombin. As shown in FIG. 6, p519 enhanced DNA synthesis over arange of concentrations while p517-520 did not. In fact, p517-520actually inhibited DNA synthesis.

EXAMPLE 5 Inhibition of Thrombin-Receptor Mediated Mitogenesis byp517-520

The observation that p517-520 inhibits alpha-thrombin stimulatedmitogenesis was somewhat startling in view of previous studiesdemonstrating that mitogenic and transmembrane signaling effects ofthrombin were not inhibited by DIP-alpha-thrombin, a thrombin derivativewhich competes for active alpha-thrombin binding. Thus, the inventorsrealized that p517-520, which is able to compete with nativealpha-thrombin for binding to high-affinity cell surface thrombinreceptors, but is unable to generate the mitogenic receptor occupancysignal, has properties not previously known in the art.

In order to explain the mechanism by which p517-520 was able to inhibitthrombin-mediated mitogenesis, the inventors measured the ability ofincreasing concentrations of alpha-thrombin to stimulate DNA synthesisin cultures to which a constant concentration (625 nM) of p517-520 hadbeen added (FIG. 7). These experiments showed that p517-520significantly shifted the dose-response curve of the cells toalpha-thrombin. For example, at two concentrations of alpha-thrombin,0.8 and 13.0 nM, DNA synthesis was inhibited by approximately 75% and35%, respectively. Thus, the inhibition of alpha-thrombin stimulation byp517-520 appears to require a 500-1000 fold molar excess of the peptide.This finding is consistent with the observation that p517-520 has alower competitive binding affinity for thrombin receptors on ME cellsthan does p508-530.

The identification of p517-520 as the high-affinity binding domain ofthrombin has several implications as to the mechanism of thrombinmitogenesis. Previous studies have demonstrated proteolytic cleavage anddisappearance of a molecule on the surface of chick embryo cells treatedwith thrombin. Cross-linking studies with active or inactive thrombinhave also identified two differently sized receptor molecules orsubstrates. The present results show that the high-affinity bindingdomain of thrombin is very close to the active-site cleft; thus, itshould be possible for thrombin to cleave its receptor. Preliminary datafrom affinity purification of the thrombin receptor supports thehypothesis that the receptor itself is proteolytically cleaved by activethrombin. It is possible that thrombin receptor occupancy may stimulatean alteration in receptor conformation necessary for the cleavage event.The present results suggest that peptides p508-530, p519-530 oralpha-thrombin itself are capable of binding to the thrombin receptor ina manner which induces such confirmational changes. In contrast,p517-520 appears to be capable only of binding to the receptor. Thus,p517-520 selectively inhibits thrombin receptor-mediated events byvirtue of its ability to selectively interact with thrombin receptors ina manner which provides the cell with a null signal.

EXAMPLE 6 Use of Stimulatory Peptides to Potentiate Cell Growth In Vitro

A number of experimental and diagnostic procedures require cells grownin vitro. Because the stimulatory peptides enhance proliferation offibroblastic cells bearing high-affinity thrombin receptors, theincorporation of such stimulatory molecules into the culture medium willprovide an effective means of potentiating cell growth. In addition,because thrombin stimulates proliferation of other cells, includingendothelial cells, these peptides may be effective in promoting growthof a number of types of cells. Use of the synthetic polypeptides asgrowth supplements has a number of advantages. It is much less expensiveto synthesize the polypeptides than its is to purify naturally occurringthrombin. Furthermore, unlike naturally occurring thrombin, thepolypeptides are relatively resistant to inhibition by serum proteaseinhibitors.

Numerous methods for preparing cells for culture are known to thoseskilled in the art. One such method, described by Carney et al. (J.Cell. Physiol., 95:13-22, 1978, incorporated herein by reference), isbelieved to be particularly well suited to the practice of this aspectof the invention.

As will be appreciated by those of skill in the art, the stimulatorypolypeptides of the present invention may be employed together with anysuitable cell culture medium to achieve the advantages of theircell-stimulation effects. For example, the present inventors have founda mixture of Dulbecco-Vogt modified Eagle's medium and Ham's F12 mediumto be a particularly appropriate base medium. To practice the invention,one adds 0.1 ug/ml-10 ug/ml of the stimulatory peptideAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO:3) to the culture medium. The cells are then incubated in anappropriate humidified atmosphere, for example, one containing 5% CO₂ inair at 37° C. At regular intervals (3 or 4 days), the spent medium isremoved from the cell culture and replaced with fresh medium formulatedas described above.

EXAMPLE 7 Treatment Protocols

Due to precautions necessarily attendant to development of every newpharmaceutical, the polypeptides of the present invention have not yetbeen tested in a clinical setting in human subjects. However, the invitro activity of these polypeptides in selectively promoting orinhibiting thrombin-mediated mitogenesis is believed to demonstrate theutility of the present invention in this regard. The following propheticembodiments represent the best mode contemplated by the presentinventors of carrying out the practice of the invention in variousclinical settings.

a. Wound Healing

It is believed that the stimulatory polypeptides will prove to be usefulin numerous clinical situations where it is desirable to potentiatewound healing. In particular, these include treatment of burn patients,those involved in severe accidents, those subjected to a variety ofsurgical procedures and those with poor wound healing responses, such asthe aged and diabetic. Although the best mode of administering thepolypeptides will depend on the particular clinical situation, it isbelieved that its administration in the form of an aerosol spray willprove to be particularly advantageous in a number of such settings.Methods for incorporating therapeutic agents into aerosol sprays arewell known in the art. Therefore, it is considered that formulation anduse of these stimulatory polypeptides in such aerosol sprays is wellwithin the skill of the art in light of the present disclosure.

The stimulatory polypeptide may also be applied to the wound in the formof a salve or lotion. Alternatively, they may be incorporated into thematerial used to dress the wound. Techniques for incorporation oftherapeutic agents compositions into salves, lotions and wound dressingsare also well known in the art and within the skill of the art in lightof the present specification.

It is believed that an effective dose of the polypeptide isapproximately between 0.5 uM-50 uM. However, exact dosages would, ofcourse, be determined empirically by experimental methods well known tothose skilled in the pharmaceutical arts.

b. Use of the Inhibitory Polypeptides

1. Inhibition of Scar Formation and Formation of Tissue Adhesions

It is further believed that the inhibitory polypeptides will proveuseful in a number of situations, for example, where inhibition offibroblast proliferation is desirable. These include prevention of scarformation and tissue adhesions.

One manner in which the invention may be practiced is by incorporatingthe inhibitory polypeptide Arg-Gly-Asp-Ala (SEQ ID NO:1) into anyvehicle suitable for application to a wound, surgical incision orsurface of a body organ. These vehicles include aerosol sprays, salvesand lotions appropriate for direct application to tissues as well assolutions appropriate for intravenous or subcutaneous injections.Methods for incorporating therapeutic agents into pharmaceuticalvehicles such as those described above is believed to be well within theskill of the art, as are methods for applying the resultantcompositions.

It is proposed that an effective dose of the polypeptide is 1 ng/cm²-10ug/cm² if the compound is applied topically. If injected, an effectivedose is that dose sufficient to obtain a concentration of thepolypeptides of from 0.1 uM to 10 uM, at the site where needed. However,exact doses, of course, should be determined by accepted pharmaceuticalmethods known to those skilled in the pharmaceutical arts.

2. Tumor Therapy

It is believed that the inhibitory polypeptides will further prove to beuseful in the treatment of various tumors, particularly in preventingmetastasis and angiogenesis. It is anticipated that the inhibitorypolypeptides could best be administered by intravenous administration.

The inhibitory polypeptides could be given daily by continuous infusionor on alternative days, with more traditional chemotherapy being givenon the intervening day. While exact doses of the inhibitory peptideswould have to be determined empirically by methods known to thoseskilled in the art, it is estimated that an effective dose would be thatamount sufficient to achieve a concentration of 0.1 uM to 10 uM at thesite where needed. Of course, as with a new pharmaceutical agent of anytype, clinical trials would be needed to establish levels at whichunacceptable toxicity would be reached.

The present invention has been disclosed in terms of specificembodiments believed by the inventor to be the best mode for carryingout the invention. However, in light of the disclosure hereby provided,those of skill in the various arts will recognize that modifications canbe made without departing from the intended scope of the invention. Forexample, any of these peptides may be administered by a number ofmethods known in the art. Furthermore, future studies are expected toresult in production of thrombin derivatives with increased stimulatoryor inhibitory activity. These and all other modifications andembodiments are intended to be within the scope of the claims.

4 4 amino acids amino acid single linear peptide 1 Arg Gly Asp Ala 1 12amino acids amino acid single linear peptide 2 Asp Ala Cys Glu Gly AspSer Gly Gly Pro Phe Val 1 5 10 23 amino acids amino acid single linearpeptide 3 Ala Gly Tyr Lys Pro Asp Glu Gly Lys Arg Gly Asp Ala Cys GluGly 1 5 10 15 Asp Ser Gly Gly Pro Phe Val 20 12 amino acids amino acidsingle linear peptide Modified-site /note= “Xaa = Ala or Ser”Modified-site /note= “Xaa = Glu or Gln” Modified-site 11 /note= “Xaa =Phe, Met, Leu, His, or Val” 4 Asp Xaa Cys Xaa Gly Asp Ser Gly Gly ProXaa Val 1 5 10

What is claimed is:
 1. A method of inhibiting tumor cell growth in apatient in need of such treatment, said method comprising the step ofadministering to the patient for a period of time and under conditionseffective to inhibit such growth a peptide comprising the tetramerArg-Gly-Asp-Ala (SEQ ID No: 1), wherein the peptide is a subsequence ofthrombin and wherein the peptide lacks a serine esterase conserveddomain.
 2. The method of claim 1 wherein the peptide is administeredintravenously.
 3. The method of claim 1 wherein the peptide is thetetramer Arg-Gly-Asp-Ala (SEQ ID NO: 1).
 4. The method of claim 3wherein the peptide is administered intravenously.
 5. A method ofinhibiting angiogenesis in a patient in need of such treatment, saidmethod comprising the step of administering a peptide to the patient fora period of time and under conditions effective for such inhibition,wherein the peptide comprises the tetramer Arg-Gly-Asp-Ala (SEQ ID No:1), wherein the peptide is a subsequence of thrombin and wherein thepeptide lacks a serine esterase conserved domain.
 6. The method of claim5 wherein the patient is in need of treatment for inhibiting tumor cellgrowth.
 7. The method of claim 6 wherein the peptide is administeredintravenously.
 8. The method of claim 6 wherein the peptide is thetetramer Arg-Gly-Asp-Ala (SEQ ID NO: 1).
 9. The method of claim 7wherein the peptide is administered intravenously.
 10. A method ofinhibiting scar formation or tissue adhesion in or on a patient in needof treatment therefor, said method comprising the step of administeringa peptide to the patient for a period of time and under conditionseffective for such inhibition, wherein the peptide comprises thetetramer Arg-Gly-Asp-Ala (SEQ ID No: 1), wherein the peptide is asubsequence of thrombin and wherein the peptide lacks a serine esteraseconserved domain.
 11. The method of claim 10 wherein the patient is inneed of treatment for a burn wound.
 12. The method of claim 11 whereinthe peptide is administered topically to the wound.
 13. The method ofclaim 10 wherein the patient is in need of treatment to inhibit tissueadhesions following thoracic surgery.
 14. The method of claim 13 whereinthe peptide is administered topically to the surface of the surgicallytreated organs.
 15. The method of claim 13 wherein the peptide isadministered intravenously.
 16. The method of claim 10 wherein thepeptide is the tetramer Arg-Gly-Asp-Ala.
 17. The method of claim 11wherein the peptide is the tetramer Arg-Gly-Asp-Ala.
 18. The method ofclaim 13 wherein the peptide is the tetramer Arg-Gly-Asp-Ala.
 19. Amethod of antagonizing thrombin receptor activity in a patient in needthereof, said method comprising the step of administering a peptide tothe patient for a period of time and under conditions effective forantagonizing such activity, wherein the peptide comprises the tetramerArg-Gly-Asp-Ala (SEQ ID No: 1), wherein the peptide is a subsequence ofthrombin and wherein the peptide lacks a serine esterase conserveddomain.
 20. The method of claim 19 wherein the peptide is the tetramerArg-Gly-Asp-Ala (SEQ ID No: 1).
 21. A method of inhibitingthrombin-mediated mitogenesis in a patient in need thereof, said methodcomprising the step of administering a peptide to the patient for aperiod of time and under conditions effective for such inhibition,wherein the peptide comprises the tetramer Arg-Gly-Asp-Ala (SEQ ID No:1), wherein the peptide is a subsequence of thrombin and wherein thepeptide lacks a serine esterase conserved domain.
 22. A method ofinhibiting thrombin-mediated fibroblast proliferation in a patient inneed of such inhibition, said method comprising the step ofadministering a peptide to the patient for a period of time and underconditions effective for such inhibition, wherein the peptide comprisesthe tetramer Arg-Gly-Asp-Ala (SEQ ID No: 1), wherein the peptide is asubsequence of thrombin and wherein the peptide lacks a serine esteraseconserved domain.